PORPHOBILINOGEN SYNTHASE FAMILY

胆色素原合酶家族

• 批准号: 6178262
• 负责人: EILEEN K JAFFE
• 金额: $ 33.42万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6178262
• 关键词: Raman spectrometry; Schiff bases; X ray crystallography; active sites; allosteric site; electron spin resonance spectroscopy; enzyme activity; enzyme biosynthesis; human tissue; ionic bond; lead; magnesium; manganese; metalloenzyme; nuclear magnetic resonance spectroscopy; porphobilinogen synthase; protein binding; protein structure function; site directed mutagenesis; zinc

DESCRIPTION: (Adapted from Applicant's Abstract) The porphobilinogen synthases (PBGS's) are ancient proteins, essential to nearly all cellular organisms. They catalyze the first common step in tetrapyrrole biosynthesis (i.e., porphyrin, chlorophyll, vitamin B12, etc.), which is the condensation of two molecules of 5-aminolevulinic acid to form porphobilinogen. To date, only one reaction intermediate is known for this complex reaction. All PBGS's are metalloenzymes and metal ion usage has suffered a unique phylogenetic switch between Zn(II) and Mg(II). Human PBGS is the primary target for lead poisoning and the less frequent of two alleles is reported to predispose humans toward this environmental disease. The proposed studies address various aspects of the PBGS's. The first specific aim uses a novel "designer gene" approach to produce and purify 100 mg quantities of human and pea PBGS because human PBGS uses Zn(II) and pea PBGS uses Mg(II). The second aim is to characterize the PBGS derived from two common human alleles and determine the molecular basis for their apparent Pb(II)-related differences. This aim includes a thorough kinetic and biochemical characterization of the human proteins, including 13C NMR and collaborative EXAFS and Raman spectroscopies and X-ray crystallography. The Raman data are complementary to the 13C NMR data on active site ligands. The third aim uses mutagenesis in conjunction with kinetics, metal and substrate binding studies, and magnetic resonance techniques, plus collaborative techniques to probe structure/function relationships in PBGS. The mutations will allow us to characterize the yet unknown intermediates in the PBGS-catalyzed reaction. Some mutations probe individual functions of the two active site Zn(II) of human PBGS and others probe the structural basis for half-sites reactivity in these homo-octameric proteins. The fourth aim focuses on the intriguing phylogenetic switch between the catalytically essential Zn(II) vs. Mg(II). Studies are directed at pea PBGS and also probe an allosteric Mg(II), absent in mammalian PBGS but present in plant and many microbial PBGS. The sum of the proposed studies is expected to yield new and important information on the mechanism of lead poisoning, on the intermediate in the PBGS catalyzed reaction, on the possible roles of both Zn(II) and Mg(II) in enzyme catalysis, on the structural basis for half sites reactivity, and on the phylogenetic differences between PBGSs. Because PBGS is an essential enzyme with distinct variations between species, we envision that a thorough understanding of the phylogenetic differences will form the basis for the rational design of a new class of organism-specific drugs.

描述：（改编自申请人的摘要）卟啉原合酶 （PBGS）是古代蛋白质，对于几乎所有细胞生物来说都是必不可少的。他们 催化四吡咯生物合成中的第一个常见步骤（即卟啉， 叶绿素，维生素B12等），这是两个分子的凝结 5-氨基乙酸以形成卟啉原。迄今为止，只有一个反应 中级以这种复杂的反应而闻名。所有PBGS都是金属酶 金属离子的使用在Zn（II）之间遭受了独特的系统发育开关 和mg（ii）。人PBG是铅中毒的主要目标，较少 据报道，经常有两个等位基因的人对此易感性 环境疾病。拟议的研究涉及 PBGS的。第一个特定目的使用新颖的“设计师基因”方法来生产 并净化100毫克的人和豌豆PBG，因为人类PBG使用 Zn（II）和PEA PBGS使用Mg（II）。第二个目的是表征PBGS 源自两个常见的人类等位基因，并确定 它们明显的PB（II）相关差异。这个目标包括一个彻底的动力学 人类蛋白质的生化特征，包括13C NMR和 协作EXAFS和RAMAN Spectroscopies和X射线晶体学。这 拉曼数据与活动现场配体的13C NMR数据互补。这 第三目的使用诱变与动力学，金属和底物结合 绑定研究和磁共振技术，以及协作 PBG中探测结构/功能关系的技术。突变 将允许我们表征未知的中间体 PBGS催化反应。某些突变探测两者的个体功能 人类PBG和其他人的主动部位Zn（II）探测了结构性基础 这些同胞聚体蛋白中的半点反应性。第四个目标重点 在催化性必不可少的系统发育开关上 Zn（ii）vs. mg（ii）。研究针对PEA PBG，也探测 变构Mg（II），哺乳动物PBG不存在，但存在于植物中，许多 微生物PBG。拟议研究的总和预计将产生新的和 关于中间体的铅中毒机理的重要信息 在PBG中催化反应，在Zn（II）和Mg（II）的可能作用上 在酶催化中，在一半站点反应性的结构基础上以及 PBGSS之间的系统发育差异。因为PBG是必不可少的酶 物种之间有明显的变化，我们设想 了解系统发育差异将构成 新型有机体特异性药物的合理设计。